In many research, diagnostic and therapeutic applications, the targets of action of a molecule, such as a reporter probe, a small molecule drug, a peptide or a nucleic acid, are intracellular. However, targeting of a molecule to selected cell types, achieving intracellular delivery to the cytosol and subsequent trafficking to the desired intracellular compartments represent challenging and elusive goals.
Significant progress in identification of cellular surface determinants, both those relatively selective for certain cell types and those that are characteristic of several or many cell types in the body, has been achieved using techniques including phage display libraries and monoclonal antibodies (Muzykantov, 2005, Expert Opinion Drug Delivery 2:909-26; Oh et al., 2004, Nature 429(6992):629-35). However, the feasibility of using these newly-identified determinants for drug delivery in humans remains to be tested. The functions of many cell surface determinants defined by these modern techniques are either not known or are responsible for vital physiological processes in the body. Thus, inadvertent intervention into or blocking of their functions may lead to harmful side effects.
Internalization of therapeutics or other molecules targeted to cells using these determinants occurs either via passive or receptor-mediated endocytosis. This endocytic internalization can be mediated by common classical endocytic pathways (e.g., clathrin- and caveolar-mediated endocytosis, macropinocytosis and phagocytosis) or by less common non-classical endocytic mechanisms (Mellman, 1992, J Exp Biol 172:39-45; Kornfeld et al., 1989, J Biol Chem 264(4):2212-20; Muro et al., 2004, Curr Vasc Pharmacol. 2(3):281-99; Riezman et al., 1997, Cell 91(6):731-8; Conner et al., 2003, Nature 422(6927):37-44). Both classical and non-classical pathways typically result in accumulation, and often degradation, of the internalized compounds in endo-lysosomal vesicles (Muro et al., 2004, Curr Vasc Pharmacol. 2(3):281-99; Riezman et al., 1997, Cell 91(6):731-8; Conner et al., 2003, Nature 422(6927):37-44).
Initially, internalized compounds traffic to early endosomes, where combined actions of H+-ATPases, Na+, K+-ATPases, and Na+/H+-exchangers (Fuchs et al., 1989, J Biol Chem 264(4):2212-20) regulate the pH to values in the interval of 6.3 to 6.5, favoring the separation of the internalized molecules from their cell receptors (Warnock, 1999, Kidney Int. 55(6):2524-5). Internalized materials then traffic to late endosomes, which have a pH around 5 to 5.5 (Mellman, 1992, J Exp Biol 172:39-45; Killisch et al, 1992, J Cell Sci. 103 (Pt 1):211-32)) and finally to lysosomes (pH 4.4 to 4.8), where acidic hydrolases lyse the internalized compounds, if these are biodegradable (Kornfeld et al., 1989, J Biol Chem 264(4):2212-20). Importantly, whether degraded or not, the materials internalized by endocytosis are confined within endo-lysosomal vesicles, which typically impedes their access to molecular targets located in the cytosol and other sub-cellular compartments.
In nature, some parasites, bacterial and viral pathogens that enter cells by classical endocytic mechanisms are able to gain access to intracellular compartments (e.g., the cytosol and, from there, the nucleus in certain cases) by escaping the endo-lysosomal vesicles in which they are contained (Cossart et al., 2004, Science 304(5668): 242-8; Campbell et al., 2005, Gene Ther. 12(18): 1353-9; Bonazzi et al., 2006, FEBS Lett 580(12):2962-7; Andrews et al., 1991, Parasitol Today 7(12):335-40). These pathogens have evolved mechanisms capable of “sensing” the decreasing pH within endosomes and lysosomes, e.g., by protonation of amphiphilic molecules which can permeate endo-lysosomal membranes.
The development of drug delivery systems that mimic the molecules and mechanism that render intracellular pathogens capable of crossing biological membranes has been pursued extensively. For instance, polycationic lipids, which are used to assist transfection of cells with DNA plasmids or small RNA molecules, bind negatively charged proteoglycans at the cell surface and thus favor cellular delivery. Disadvantageously, however, polycationic lipids form holes in the plasma membrane which causes cellular damage and death (Singh et al., 2004, Chem. Biol. 11(5):713-23; Dincer et al., 2005, Gene Ther. 12 Suppl 1:S139-45; Zuhorn et al., 2005, Mol. Ther. 11(5):801-10). In a related example, positively charged, arginine rich cell penetrating peptides (e.g., RGD and Tat) bind to the cell surface by electrostatic interactions and have been reported to facilitate subsequent intracellular delivery of conjugated cargoes (Melikov et al., 2005, Cell Mol Life Sci. 62(23):2739-49; Magzoub et al., 2005, Biochemistry 44(45): 14890-7; Suk et al., 2006, Biomaterials). However, it is remains uncertain whether these conjugates can cross the plasma membrane or are internalized within endo-lysosomal vesicles via natural endocytic pathways. Fusogenic peptides derived from bacterial toxins (e.g., hemagglutinin-derived, GALA peptides) are believed to induce formation of holes in the endosomal membrane upon internalization, due to changes in the peptide structure (e.g., from random coils to amphiphilic helixes capable of penetrate the endosomal membrane (Kakudo et al., 2004, Biochemistry 43(19):5618-28)), which occur in response to gradual pH lowering in these vesicular compartments. However, such fusogenic peptides, which have also been shown to permeate the plasma membrane (and therefore cause adverse effects including cell damage and death), appear to be effective only when presented to the cell as part of a phospholipid carriers, e.g., liposomes, which have formidable intrinsic limitations for delivery of many types of cargoes (e.g., proteins) and also have very limited circulation time in the bloodstream (Kakudo et al., 2004, Biochemistry 43(19):5618-28).
Other state-of-the-art means designed to overcome endosomal membranes without affecting the plasma membrane include polymer carriers that are sensitive to pH and temperature-responsive polyelectrolyte hydrogels (Yessine et al., 2004, Adv Drug Deliv Rev. 56(7):999-1021; Choi et al., 2006, Biomacromolecules 7(6): 1864-7015-26; Oishi et al., 2006, Bioconjug Chem. 17(3):677-88; Stayton et al., 2005, Orthod Craniofac Res. 8(3):2 19-25). Polymer carriers that can sense pH (e.g., acrylic acid derivatives) basically act as pH buffers and only become protonated when the endosomal pH decreases. However, currently available carriers with these characteristics render monomer non-biodegradable and are likely to be cytotoxic (Yessine et al., 2004, Adv Drug Deliv Rev. 56(7):999-1021; Oishi et al., 2006, Bioconjug Chem. 17(3):677-88; Stayton et al., 2005, Orthod Craniofac Res. 8(3):2 19-25). Permeating activity of temperature-responsive polyelectrolyte hydrogel carriers can be controlled after their internalization by varying the hydrogel hydration rate in a temperature dependent manner, which leads to changes in the carrier volume and results in lysis of the endo-lysosomal vesicle (Choi et al., 2006, Biomacromolecules 7(6): 1864-70). This procedure requires changes in temperature, which can be applied only to focal targets with well-known localization, hence disseminated targets and targets with unknown localization cannot be treated. This method is also highly invasive, which greatly restricts applications of this type of carriers. Finally, targeting of such types of nano-scale carriers in the vasculature remains to be designed and tested.
Thus, there is a need in the art for a method of delivering exogenous material into cells. The present invention addresses this need.